Effect of acid/base balance on H-ATPase 31 kD subunit mRNA levels in collecting duct cells.

ABSTRACT

The cortical collecting duct (CCD) adapts to disturbances of acid/base balance by adjusting the direction and magnitude of its HCO3 transport. The molecular events involved in this adaptation are incompletely understood, but it seems that adaptation is accompanied by changes in the activity and intracellular distribution of the vacuolar H-ATPase. The goal of this study was to examine the effects of metabolic acidosis and alkali load on the expression of the mRNA encoding the 31 kD subunit of the vacuolar H-ATPase in rabbit CCD cells. Pairs of rabbits received either a NH4Cl load or a NaHCO3 load for 16 hours, resulting in a urinary pH of 5.53 +/- 0.38 and 8.42 +/- 0.10, respectively. CCD cells were isolated by immunodissection and mRNA levels of the H-ATPase 31 kD subunit and of beta-actin were determined from the same cDNA samples by quantitative RT-PCR. H-ATPase mRNA levels were significantly higher in CCD cells from acidotic than alkali-loaded rabbits (2.51 +/- 1.3 vs. 0.65 +/- 0.2; P < 0.05). Similar differences in the H-ATPase 31 kD subunit mRNA levels were observed by Northern blotting. beta-actin mRNA levels were comparable in CCD cells of the two groups. The distribution of the H-ATPase 31 kD subunit mRNA was determined among the three cell types of the CCD, that is in alpha- and beta-intercalated cells (alpha-ICC and beta-ICC) and principal cells (PC) isolated by fluorescence-activated cell sorting. The level of expression was comparable in alpha-ICCs and beta-ICCs, whereas PCs contained very low levels of H-ATPase mRNA. In both alpha-ICC and beta-ICC the levels of the 31 kD H-ATPase mRNA were significantly higher in acidotic than in alkali-loaded rabbits. These results indicate that in the rabbit CCD changes in acid/base balance not only regulate the subcellular distribution of the vacuolar H-ATPase but also alter its expression, at least at the mRNA level.